During animal development, the various mechanisms that determine cell fates must be well coordinated. For example, long-range diffusible signaling molecules, short-range cell surface signaling molecules, and nuclear transcription factors must all function together as an integrated system. Further, these mechanisms must also operate in the context of a growing and continually changing tissue. The long-term goal of this project is to understand, in molecular terms, how such different mechanisms are integrated with each other, and with developmental time. The control of developmental pathways by the Hox genes provides an example of this problem. The Hox genes all encode DNA-binding homeodomain proteins that regulate the transcription of specific sets of downstream, target genes. Hox proteins, however, do not act alone: they bind DNA together with co-factor that increase both their binding site specificity and affinity. One well characterized family of Hox co-factors are encoded by the Drosophila extradenticle and vertebrate pbx genes. Unlike most homeodomain proteins, the Extradenticle protein is post-translationally regulated by controlling its cytoplasmic-to-nuclear translocation. Another homeodomain protein, encoded by the Drosophila homothorax gene, is required for Extradenticle's nuclear localization. In some cells, the nuclear localization of Extradenticle is controlled by diffusible signaling molecules, which, therefore, indirectly control Hox function. The work proposed is divided into four parts: the first investigates how Homothorax controls Extradenticle's nuclear localization; the second investigates Homothorax's role as a DNA-binding co-factor for the Hox proteins; and the third investigates a role for homothorax as a selector gene for antennal development. The four part proposes to use a genetic screen to identify genes that modulate Homothorax function. Parts one and two use cell biology to investigate the mechanism of Exd nuclear localization, and biochemistry to characterize critical protein-protein and protein-DNA interactions. The third and fourth parts use Drosophila genetics to investigate these problems. Characterizing these developmental mechanisms will shed light on how the vertebrate versions of these proteins, when altered, can cause cancer and human birth defects.